There is a continuing need in medical practice and research, and in analytical and diagnostic procedures, for rapid and accurate determinations of chemical and biological substances present in various fluids, such as biological fluids. For example, the presence of drugs, narcotics, hormones, proteins, toxins, microorganisms, viruses, steroids or nucleic acids must be rapidly and accurately detected for effective research, diagnosis or treatment of various diseases or conditions.
Prostaglandin E.sub.2 (PGE.sub.2) is a potent biochemical mediator of inflammation in the body. Elevated levels of PGE.sub.2 in crevicular fluid have been shown to be an indicator of periodontal disease. Due to the cyclical nature of periodontal diseases, an accurate measure of disease activity would be useful to determine periods of disease exacerbation and to aid in favorable disease treatment. If PGE.sub.2 can be detected at very low concentrations (100 nmolar or less), the clinical usefulness of PGE.sub.2 as an indicator of periodontal disease is optimized.
A wide variety of analytical methods have been developed in recent decades to detect various chemical or biological substances. Most of such methods rely on what are known in the art as "specific binding" reactions in which an unknown substance to be detected (known as a "specific binding ligand") reacts specifically and preferentially with a corresponding "receptor" molecule. Most well known specific binding reactions occur between immunoreactants, such as antibodies and antigens (foreign substances which produce immunological responses), but other specific binding reactions (such as avidin with biotin or a sugar with a lectin) are also known.
Many of the assay formats known in the art require one or more of the reactants to be immobilized on a solid substrate so that nonimmobilized reactants can be separated from immobilized reactants. "Sandwich" and "direct binding assays" are some of the assay formats which have been used in the art which require separation steps.
The standard immunological techniques, however, are more difficult to use with success in the detection of low molecular weight target specific binding ligands, such as PGE.sub.2. It is particularly desirable to detect low concentrations (100 nmolar or less) of such ligands, but the standard immunological techniques are not always dependable for this purpose.
Quantification of low molecular weight target specific binding ligands generally involves a competition between unlabeled ligand (in a test sample) and labeled ligand for the binding sites of a receptor for the ligand. This is a typical competitive specific binding assay. Ligand in the test sample displaces labeled ligand from the receptor, and the amount of displacement is quantified by various methods. This results in an inverse correlation between the amount of bound ligand and the generated signal. Measurement of unbound ligand will yield a positive correlation.
Generally, the signal from bound ligand is easier to measure, but the signal is more difficult to interpret by health practitioners where there is a lack of sophisticated diagnostic training and equipment (for example, in doctors' offices).
Another problem with standard competitive specific binding assays is that the measurement of the free or bound labeled ligand is limited by the efficiency of the separation of free and bound molecules. If the free labeled ligand is not successfully separated from the bound labeled ligand, the sensitivity and specificity of the assay will be adversely affected.
Thus, there is a need for a sensitive specific binding assay in which efficient separation of free and bound labeled ligand is achieved to rapidly and easily detect low concentrations of various target specific binding ligands (especially those of low molecular weight).